Neuroproteomics — LC-MS Quantitative Approaches

Neuroproteomics is a scientific field that aims to study all the proteins of the central nervous system, their expression, function, and interactions. The central nervous system is intricate and heterogeneous, and the study of its proteome is consequently complex, with many biological questions still requiring deep investigation. For this, mass spectrometry approaches, most often coupled with liquid chromatography (LC-MS), have been the number one choice in proteomics, and over the years it has added many important findings to the field. At this point it is important that proteomics turns to the quantitative expression of proteins instead of only identifying which proteins are present in a given sample, much because the most important alterations may be slight alterations in the quantity of a protein in a given situation. Therefore, many LC-MS quantitative approaches have been developed relying on the labeling of the proteins or even by using label-free techniques

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology

Background: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. Methods: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. Results: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Conclusions: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.The authors would like to acknowledge the funding agencies that supported this work: Fundacao para a Ciencia e Tecnologia (FCT), Portugal, projects reference: PTDC/SAU-GMG/113795/2009 (BMC); SFRH/BD/88121/2012 (JVdC); SFRH/BD/103075/2014 (EDG); IF/00601/2012 (BMC); IF/00111/2013 (AJS); SFRH/BD/81495/2011 (SIA); PTDC/NEU-NMC/0205/2012, PTDC/NEUSCC/ 7051/2014, PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013 (BM); Fundacao Calouste Gulbenkian (BMC); Liga Portuguesa Contra o Cancro (BMC); " COMPETE Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER-Fundo Europeu de Desenvolvimento Regional) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005; FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038; and project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funding body did not have a role in the design of the study, in collection, analysis or interpretation of data, or in writing the manuscript

Crosstalk between glial and glioblastoma cells triggers the "go-or-grow" phenotype of tumor cells

Background: Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells. Methods: Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways. Results: The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Conclusions: Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the "go-or-grow" phenotypic switch of GBM cells.Fundação para a Ciência e Tecnologia (IF/00601/2012 to B.M.C.; IF/00111 to A.J.S; SFRH/BD/52287/2013 to A.I.O.; SFRH/BD/81495/2011 to S.I.A.; SFRH/BD/88121/2012 to J.V.C.; projects PTDC/SAU-GMG/113795/2009 to B.M.C.; PTDC/NEU-NMC/0205/2012, PTDC/NEU-SCC/7051/2014, PEst-C/SAU/LA0001/2013–2014 and UID/NEU/04539/2013 to B.M.), Liga Portuguesa Contra o Cancro (B.M.C.), Fundação Calouste Gulbenkian (B.M.C.) and Inter-University Doctoral Programme in Ageing and Chronic Disease (PhDOC; to A.I.O.). Project co-financed by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER), Programa Operacional Factores de Competitividade (COMPETE), and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005info:eu-repo/semantics/publishedVersio

Metabolic Priming as a Tool in Redox and Mitochondrial Theragnostics

Theragnostics is a promising approach that integrates diagnostics and therapeutics into a single personalized strategy. To conduct effective theragnostic studies, it is essential to create an in vitro environment that accurately reflects the in vivo conditions. In this review, we discuss the importance of redox homeostasis and mitochondrial function in the context of personalized theragnostic approaches. Cells have several ways to respond to metabolic stress, including changes in protein localization, density, and degradation, which can promote cell survival. However, disruption of redox homeostasis can lead to oxidative stress and cellular damage, which are implicated in various diseases. Models of oxidative stress and mitochondrial dysfunction should be developed in metabolically conditioned cells to explore the underlying mechanisms of diseases and develop new therapies. By choosing an appropriate cellular model, adjusting cell culture conditions and validating the cellular model, it is possible to identify the most promising therapeutic options and tailor treatments to individual patients. Overall, we highlight the importance of precise and individualized approaches in theragnostics and the need to develop accurate in vitro models that reflect the in vivo conditions

Modulation of signaling pathways by DJ-1: An updated overview

Efforts have been made to understand the physiological and pathological role of DJ-1, a Parkinson's disease (PD)-associated protein, to provide new insights into PD pathophysiology. Such studies have revealed several neuroprotective roles of DJ-1, from which its ability to modulate signaling pathways seems to be of utmost importance for cell death regulation by DJ-1. Indeed, research on these topics has led to a higher number of publications disclosing a variety of mechanisms through which DJ-1 is able to modulate signaling pathways in distinct disease-related contexts. Thus, this graphical review presents the most relevant findings concerning the mechanisms through which DJ-1 exerts its regulatory activity on signaling cascades relevant for DJ-1 neuroprotective action, namely ERK1/2, PI3K/Akt, and ASK1 pathways, and Nrf2 and p53 transcription factors-related signaling. A greater focus was given to perform an overview of the research interests over the last years, especially in the most recent works, to highlight the current research lines in this topic, and point out future directions in the field. In addition, the impact of DJ-1 mutations causative of PD and the importance of the redox status of DJ-1's cysteine residues for the action of DJ-1 on signaling modulation was also addressed to uncover the potential pathological mechanisms associated with loss of DJ-1 native function

Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis

The pinewood nematode, Bursaphelenchus xylophilus, recognized as a worldwide major forest pest, is a migratory endoparasitic nematode with capacity to feed on pine tissues and also on fungi colonizing the trees. Bursaphelenchus mucronatus, the closest related species, differs from B. xylophilus on its pathogenicity, making this nematode a good candidate for comparative analyses. Secretome profiles of B. xylophilus and B. mucronatus were obtained and proteomic differences were evaluated by quantitative SWATH-MS. From the 681 proteins initially identified, 422 were quantified and compared between B. xylophilus and B. mucronatus secretomes and from these, 243 proteins were found differentially regulated: 158 and 85 proteins were increased in B. xylophilus and B. mucronatus secretomes, respectively. While increased proteins in B. xylophilus secretome revealed a strong enrichment in proteins with peptidase activity, the increased proteins in B. mucronatus secretome were mainly related to oxidative stress responses. The changes in peptidases were evaluated at the transcription level by RT-qPCR, revealing a correlation between the mRNA levels of four cysteine peptidases with secretion levels. The analysis presented expands our knowledge about molecular basis of B. xylophilus and B. mucronatus hosts interaction and supports the hypothesis of a key role of secreted peptidases in B. xylophilus pathogenicity

Comparative Analysis of Bursaphelenchus xylophilus Secretome Under Pinus pinaster and P. pinea Stimuli

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the pine wilt disease's (PWD) causal agent, is a migratory endoparasitic nematode skilled to feed on pine tissues and on fungi that colonize the trees. In order to study B. xylophilus secretomes under the stimulus of pine species with different susceptibilities to disease, nematodes were exposed to aqueous pine extracts from Pinus pinaster (high-susceptible host) and P. pinea (low-susceptible host). Sequential windowed acquisition of all theoretical mass spectra (SWATH-MS) was used to determine relative changes in protein amounts between B. xylophilus secretions, and a total of 776 secreted proteins were quantified in both secretomes. From these, 22 proteins were found increased in the B. xylophilus secretome under the P. pinaster stimulus and 501 proteins increased under the P. pinea stimulus. Functional analyses of the 22 proteins found increased in the P. pinaster stimulus showed that proteins with peptidase, hydrolase, and antioxidant activities were the most represented. On the other hand, gene ontology (GO) enrichment analysis of the 501 proteins increased under the P. pinea stimulus revealed an enrichment of proteins with binding activity. The differences detected in the secretomes highlighted the diverse responses from the nematode to overcome host defenses with different susceptibilities and provide new clues on the mechanism behind the pathogenicity of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD024011

Proteomic Analyses Reveal New Insights on the Antimicrobial Mechanisms of Chitosan Biopolymers and Their Nanosized Particles against Escherichia coli

The well-known antimicrobial effects of chitosan (CS) polymers make them a promising adjuvant in enhancing antibiotic effectiveness against human pathogens. However, molecular CS antimicrobial mechanisms remain unclear, despite the insights presented in the literature. Thus, the aim of the present study was to depict the molecular effects implicated in the interaction of low or medium molecular mass CS polymers and their nanoparticle-counterparts against Escherichia coli. The differential E. coli proteomes sensitized to either CS polymers or nanoparticles were investigated by nano liquid chromatography-mass spectrometry (micro-LC-MS/MS). A total of 127 proteins differentially expressed in CS-sensitized bacteria were predominantly involved in (i) structural functions associated to the stability of outer membrane, (ii) increment of protein biosynthesis due to high abundance of ribosomal proteins and (iii) activation of biosynthesis of amino acid and purine metabolism pathways. Antibacterial activity of CS polymers/nanoparticles seems to be triggered by the outer bacterial membrane disassembly, leading to increased protein biosynthesis by diverting the metabolic flux to amino acid and purine nucleotides supply. Understanding CS-antibacterial molecular effects can be valuable to optimize the use of CS-based nanomaterials in food decontamination, and may represent a breakthrough on CS nanocapsules-drug delivery devices for novel antibiotics, as the chitosan-disassembly of bacteria cell membranes can potentialize antibiotic effects.This research was funded by Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro—FAPERJ Grants: Pos-doc Nota 10 program, PDR-10-E-26/202.319/2017; CNE, E-26/203.039/2015; CNE, E-26/202.815/2018; Sediadas E-26/010002864/2014 and Nottinghan/Birminghan, E-26/010.002673/2014. This study was partially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI), by ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI—“Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa 2020” and by national funds through “FCT—Fundação para a Ciência e a Tecnologia”. This work was partially supported by: by the European Regional Development Fund (ERDF) through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., OE FCT/MCTES (PIDDAC) under projects: POCI-01-0145-FEDER-007440 (strategic project UID/NEU/04539/2019) and POCI-01-0145-FEDER-029311 (ref.: PTDC/BTM-TEC/29311/2017)

Defective mitochondria‐lysosomal axis enhances the release of extracellular vesicles containing mitochondrial DNA and proteins in Huntington's disease

Abstract Mitochondrial and autophagy dysfunction are mechanisms proposed to be involved in the pathogenesis of several neurodegenerative diseases. Huntington's disease (HD) is a progressive neurodegenerative disorder associated with mutant Huntingtin‐induced abnormalities in neuronal mitochondrial dynamics and quality control. Former studies suggest that the removal of defective mitochondria may be compromised in HD. Mitochondrial quality control (MQC) is a complex, well‐orchestrated pathway that can be compromised through mitophagy dysregulation or impairment in the mitochondria‐lysosomal axis. Another mitochondrial stress response is the generation of mitochondrial‐derived vesicles that fuse with the endolysosomal system and form multivesicular bodies that are extruded from cells as extracellular vesicles (EVs). In this work, we aimed to study the presence of mitochondrial components in human EVs and the relation to the dysfunction of both mitochondria and the autophagy pathway. We comprehensively characterized the mitochondrial and autophagy alterations in premanifest and manifest HD carriers and performed a proteomic and genomic EVs profile. We observed that manifest HD patients exhibit mitochondrial and autophagy impairment associated with enhanced EVs release. Furthermore, we detected mitochondrial DNA and proteins in EVs released by HD cells and in neuronal‐derived EVs including VDAC‐1 and alpha and beta subunits of ATP synthase F1. HD‐extracellular vesicles transport higher levels of mitochondrial genetic material in manifest HD patients, suggesting an alternative pathway for the secretion of reactive mitochondrial components. This study provides a novel framework connecting EVs enhanced release of mitochondrial components to mitochondrial and lysosomal dysfunction in HD